The present invention relates generally to methods for modulating desmosomal cadherin-mediated functions, and more particularly to the use of modulating agents derived from desmocollin and desmoglein cell adhesion recognition sequences for inhibiting or enhancing functions mediated by such desmosomal cadherins.
Cadherins are a rapidly expanding superfamily of calcium-dependent cell adhesion molecules (CAMs) (for review, see Munro et al., In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34, RG Landes Co., Austin Tex., 1996). All cadherins appear to be membrane glycoproteins that generally promote cell adhesion through homophilic interactions (a cadherin on the surface of one cell binds to an identical cadherin on the surface of another cell), although cadherins also appear to be capable of forming heterotypic complexes with one another under certain circumstances and with lower affinity.
There are many different types of cadherins. The most extensively studied group of cadherins is known as the classical, or type I, cadherins. Classical cadherins have been shown to regulate epithelial, endothelial, neural and cancer cell adhesion, with different cadherins expressed on different cell types. All classical cadherins have a similar structure. As illustrated in FIG. 1A, classical cadherins are composed of five extracellular domains (EC1-EC5), a single hydrophobic domain (TM) that transverses the plasma membrane (PM), and two cytoplasmic domains (CP1 and CP2). The calcium binding motifs DXNDN (SEQ ID NO:1), DXD and LDRE (SEQ ID NO:2) are interspersed throughout the extracellular domains, and each 110 amino acid region that contains such motifs is considered a cadherin repeat. The first extracellular domain (EC1) contains the cell adhesion recognition (CAR) sequence, HAV (His-Ala-Val), along with flanking sequences on either side of the CAR sequence that play a role in conferring specificity. Synthetic peptides containing the HAV sequence and antibodies directed against such peptides have been shown to inhibit classical cadherin-dependent processes (Munro et al., supra; Blaschuk et al., J. Mol. Biol. 211:679-82, 1990; Blaschuk et al., Develop. Biol. 139:227-29, 1990; Alexander et al., J. Cell. Physiol 156:610-18, 1993).
Cadherins that contain calcium binding motifs within extracellular domain cadherin repeats, but do not contain an HAV CAR sequence, are considered to be nonclassical cadherins (illustrated in FIGS. 1B to 1AA). To date, nine groups of nonclassical cadherins have been identified (types II-X). These cadherins are also membrane glycoproteins. Type II, or atypical, cadherins include OB-cadherin (cadherin-11; see Getsios et al., Developmental Dynamics 211:238-247, 1998; Simonneau et al., Cell Adhesion and Communication 3:115-130, 1995; Okazaki et al., J. Biological Chemistry 269:12092-12098, 1994), cadherin-5 (VE-cadherin; see Navarro et al., J. Cell Biology 140:1475-1484, 1998), cadherin-6 (K-cadherin; see Shimoyama et al., Cancer Research 55:2206-2211, 1995; Shimazui et al., Cancer Research 56:3234-3237, 1996; Inoue et al., Developmental Dynamics 211:338-351, 1998; Getsios et al., Developmental Dynamics 211:238-247, 1998), cadherin-7 (see Nakagawa et al., Development 121:1321-1332, 1995), cadherin-8 (see Suzuki et al., Cell Regulation 2:261-270, 1991), cadherin-12 (Br-cadherin; see Tanihara et al., Cell Adhesion and Communication 2:15-26, 1994), cadherin-14 (see Shibata et al., J. Biological Chemistry 272:5236-5240, 1997), cadherin-15 (M-cadherin; see Shimoyama et al., J. Biological Chemistry 273:10011-10018, 1998), and PB-cadherin (see Sugimoto et al., J. Biological Chemistry 271:11548-11556, 1996). For a general review of atypical cadherins, see Redies and Takeichi, Developmental Biology 180:413-423, 1996 and Suzuki et al., Cell Regulation 2:261-270, 1991.
Types III-X include LI-cadherin (type III; see Berndorff et al., J. Cell Biology 125:1353-1369, 1994), T-cadherin (type IV; see Ranscht, U.S. Pat. No. 5,585,351; Tkachuk et al., FEBS Lett. 421:208-212, 1998; Ranscht et al., Neuron 7:391-402, 1991; Sacristan et al., J. Neuroscience Research 34:664-680, 1993; Vestal and Ranscht, J. Cell Biology 119:451-461, 1992; Fredette and Ranscht, J. Neuroscience 14:7331-7346, 1994; Ranscht and Bronner-Fraser, Development 111:15-22, 1991), protocadherins (type V; e.g., protocadherins 42, 43 and 68; see Sano et al., EMBO J. 12:2249-2256, 1993; GenBank Accession Number AF029343), desmocollins (type VI; e.g., desmocollins 1, 2, 3 and 4; see King et al., Genomics 18:185-194, 1993; Parker et al., J. Biol Chem. 266:10438-10445, 1991; King et al., J. Invest. Dermatol 105:314-321, 1995; Kawamura et al., J. Biol Chem. 269:26295-26302, 1994), desmogleins (type VII; e.g., desmogleins 1 and 2; see Wheeler et al., Proc. Natl Acad. Sci. USA 88:4796-4800; Koch et al., Eur. J. Cell. Biol 55:200-208, 1991), and cadherin-related neuronal receptors (type X; see Kohmura et al., Neuron 20:1137-1151, 1998).
Most studies of nonclassical cadherins have focused on atypical or type II cadherins. The structure of such cadherins is similar to that of the type I cadherins, but they do not contain the CAR sequence, HAV (FIG. 1B). Atypical cadherins appear to mediate a wide variety of functions. Additional variation is seen in the structures of types III-X cadherins. Although less studied, such cadherins also appear to play a role in diverse functions. Desmosomal cadherins, for example, are present within desmosomes, the intercellular junctions that provide adhesion and membrane anchors for the intermediate filament cytoskeleton. These cadherins (e.g., desmogleins and desmocollins) play a role in desmosomal adhesion, which is critically important for normal tissue construction and epidermis structure. Abnormal desmosomes appear in certain types of carcinomas and other skin disorders (see Chidgey, Histol Histopathol. 12:1159-1168, 1997).
Notwithstanding these recent advances, nonclassical cadherin function remains poorly understood at the biological and molecular levels. Accordingly, there is a need in the art for identifying sequences involved in modulating nonclassical cadherin-dependent functions, such as cell adhesion mediated by desmosomal cadherins, and for the development of methods employing such sequences to inhibit processes such as cell adhesion. The present invention fulfills these needs and further provides other related advantages.
Briefly stated, this invention provides compositions and methods for modulating desmosomal cadherin-mediated functions, such as cell adhesion. Within certain aspects, modulating agents are provided. Such agents are capable of modulating (i.e., inhibiting or enhancing) one or more functions mediated by the desmosomal cadherins desmoglein (dsg) or desmocollin (dsc). A modulating agent may comprise at least one of: (a) a native dsc or dsg CAR sequence; (b) an analogue of such a CAR sequence that is capable of modulating dsc- or dsg-mediated cell adhesion; (c) a non-peptide peptidomimetic of a dsg or dsg CAR sequence that is capable of modulating dsc- or dsg-mediated cell adhesion; (d) an antibody, or antigen-binding fragment thereof, that specifically binds a dsc or dsg CAR sequence; and/or (e) a polynucleotide encoding a native dsc or dsg CAR sequence or analogue thereof that is capable of modulating dsc- or dsg-mediated cell adhesion. Certain preferred modulating agents comprise a desmoglein-1, desmoglein-2, desmoglein-3, desmocollin-1, desmocoffin-2, desmocollin-3 or desmocollin-4 CAR sequence, or an analogue of any of the foregoing CAR sequences. An analogue is generally a peptide sequence that is at least 50% identical to a dsg or dsc CAR sequence, and (i) detectably inhibits a function that is modulated by the dsg or dsc; or (ii) detectably enhances adhesion of cells that express the dsg or dsc. Modulating agents preferably contain no more than 85, and preferably no more than 50, consecutive amino acid residues present within the dsg or dsc. Certain modulating agents comprise a dsg or dsc CAR sequence and are 3-16 amino acid residues in length. Within certain specific embodiments, a modulating agent as described above is a peptide ranging in size from 3 to 50, preferably from 4 to 16, amino acid residues.
For certain modulating agents, the dsg or dsc CAR sequence has the formula:
Aaa-Phe-Baa-Ile/Leu/Val-Asp/Asn/Glu-Caa-Daa-Ser/Thr/Asn-Glyxe2x80x83xe2x80x83(SEQ ID NO:3)
wherein Aaa, Baa, Caa and Daa are independently selected amino acid residues; Ile/Leu/Val is an amino acid that is selected from the group consisting of isoleucine, leucine and valine, Asp/Asn/Glu is an amino acid that is selected from the group consisting of aspartate, asparagine and glutamate; and Ser/Thr/Asn is an amino acid that is selected from the group consisting of serine, threonine or asparagine. For other modulating agents as described above, the dsg or dsc CAR sequence consists of at least three consecutive amino acid residues, and preferably at least five consecutive amino acid residues, of a region of the dsg or dsc having the formula recited above.
Within certain specific embodiments, a modulating agent as provided herein comprises: (a) one or more desmoglein CAR sequences selected from the group consisting of NQK, NQKT (SEQ ID NO:63), NQKTG (SEQ ID NO:64), INQK (SEQ ID NO:65), INQKT (SEQ ID NO:66), INQKTG (SEQ ID NO:67), VINQK (SEQ ID NO:68), VINQKT (SEQ ID NO:69), VINQKTG (SEQ ID NO:70), FVINQK (SEQ ID NO:71), FVINQKT (SEQ ID NO:72), FVINQKTG (SEQ ID NO:73), IFVINQK (SEQ ID NO:74), IFVINQKT (SEQ ID NO:75), IFVINQKTG (SEQ ID NO:76), NRN, NRNT (SEQ ID NO:77), NRNTG (SEQ ID NO:78), INRN (SEQ ID NO:79), INRNT (SEQ ID NO:80), INRNTG (SEQ ID NO:81), IINRN (SEQ ID NO:82), IINRNT (SEQ ID NO:83), IINRNTG (SEQ ID NO:84), FIINRN (SEQ ID NO:85), FIINRNT (SEQ ID NO:86), FIINRNTG (SEQ ID NO:87), MFIINRN (SEQ ID NO:88), MFIINRNT (SEQ ID NO:89), MFIINRNTG (SEQ ID NO:90), NKD, NKDT (SEQ ID NO:91), NKDTG (SEQ ID NO:92), LNKD (SEQ ID NO:93), LNKDT (SEQ ID NO:94), LNKDTG (SEQ ID NO:95), YLNKD (SEQ ID NO:96), YLNKDT (SEQ ID NO:97), YLNKDTG (SEQ ID NO:98), FYLNKD (SEQ ID NO:99), FYLNKDT (SEQ ID NO:100), FYLNKDTG (SEQ ID NO:101), VFYLNKD (SEQ ID NO:102), VFYLNKDT (SEQ ID NO:103) and VFYLNKDTG (SEQ ID NO:104); or (b) an analogue of any of the foregoing sequences that differs in one or more substitutions, deletions, additions and/or insertions such that that ability of the analogue to modulate a desmoglein-mediated function is not substantially diminished. For example, the agent may comprise a linear peptide having the sequence Nxe2x80x94Ac-IFVINQKTG-NH2 (SEQ ID NO:105), Nxe2x80x94Ac-MFIINRNTG-NH2 (SEQ ID NO:106) or Nxe2x80x94Ac-VFYLNKDTG-NH2 (SEQ ID NO:107). The desmoglein CAR sequence may, but need not, be present within a cyclic peptide.
Within other specific embodiments, a modulating agent as provided herein may comprise: (a) one or more desmocollin CAR sequences selected from the group consisting of EKD, EKDT (SEQ ID NO:108), EKDTG (SEQ ID NO:109), IEKD (SEQ ID NO:110), IEKDT (SEQ ID NO:111), IEKDTG (SEQ ID NO:112), YIEKD (SEQ ID NO:113), YIEDT (SEQ ID NO:114), YIEKDTG (SEQ ID NO:115), FYIEKD (SEQ ID NO:116), FYIEKDT (SEQ ID NO:117), FYIEKDTG (SEQ ID NO:118), LFYIEKD (SEQ ID NO:119), LFYIEKDT (SEQ ID NO:120), LFYIEKDTG (SEQ ID NO:121), ERD, ERDT (SEQ ID NO:122), ERDTG (SEQ ID NO:123), VERD (SEQ ID NO:124), VERDT (SEQ ID NO:125), VERDTG (SEQ ID NO:126), YVERD (SEQ ID NO:127), YVERDT (SEQ ID NO:128), YVERDTG (SEQ ID NO:129), FYVERD (SEQ ID NO:130), FYVERDT (SEQ ID NO:131), FYVERDTG (SEQ ID NO:132), LFYVERD (SEQ ID NO:133), LFYVERDT (SEQ ID NO:134), LFYVERDTG (SEQ ID NO:135), IERD (SEQ ID NO:136), IERDT (SEQ ID NO:137), IERDTG (SEQ ID NO:138), YIERD (SEQ ID NO:139), YIERDT (SEQ ID NO:140), YIERDTG (SEQ ID NO:141), FYIERD (SEQ ID NO:142), FYIERDT (SEQ ID NO:143), FYIERDTG (SEQ ID NO:144), LFYIERD (SEQ ID NO:145), LFYIERDT (SEQ ID NO:146) and LFYIERDTG (SEQ ID NO:147); or (b) an analogue of any of the foregoing sequences that differs in one or more substitutions, deletions, additions and/or insertions such that that ability of the analogue to modulate a desmocollin-mediated function is not substantially diminished. For example, the agent may comprise a linear peptide having the sequence Nxe2x80x94Ac-LFYIEKDTG-NH2 (SEQ ID NO:148), Nxe2x80x94Ac-LFYVERDTG-NH2 (SEQ ID NO:149) or Nxe2x80x94Ac-LFYIERDTG-NH2 (SEQ ID NO:150). The desmocollin CAR sequence may, but need not, be present within a cyclic peptide.
Within other specific embodiments, a modulating agent as provided herein may comprise: (a) one or more Desmoglein-1 CAR sequences selected from the group consisting of: RAL, RALN (SEQ ID NO:151), RALNS (SEQ ID NO:152, RALNSM (SEQ ID NO:153), RALNSL (SEQ ID NO:154), RALNSMG (SEQ ID NO:155), RALNSLG (SEQ ID NO:156), CRAL (SEQ ID NO:157), CRALN (SEQ ID NO:158), CRALNS (SEQ ID NO:159), CRALNSM (SEQ ID NO:160), CRALNSL (SEQ ID NO:161), CRALNSMG (SEQ ID NO:162), CRALNSLG (SEQ ID NO:163), YCRAL (SEQ ID NO:164), YCRALN (SEQ ID NO:165), YCRALNS (SEQ ID NO:166), YCRALNSM (SEQ ID NO:167), YCRALNSL (SEQ ID NO:168), YCRALNSMG (SEQ ID NO:169), YCRALNSLG (SEQ ID NO:170),IYRAL (SEQ ID NO:171), IYRALN (SEQ ID NO:172), IYRALNS (SEQ ID NO:173), IYRALNSM (SEQ ID NO:174), IYRALNSL (SEQ ID NO:175), IYRALNSMG (SEQ ID NO:176) or IYRALNSLG (SEQ ID NO:177); or (b) an analogue of any of the foregoing sequences that differs in one or more substitutions, deletions, additions and/or insertions such that that ability of the analogue to modulate a desmoglein-mediated function is not substantially diminished. Linear peptides having such sequences may be modified at the N- and/or C-termini, as in the peptides Nxe2x80x94Ac-IYRALNSMG-NH2 (SEQ ID NO:178) and Nxe2x80x94Ac-IYRALNSLG-NH2 (SEQ ID NO:179).
Within other specific embodiments, a modulating agent as provided herein may comprise: (a) one or more Desmoglein-2 CAR sequences selected from the group consisting of: YAL, YALD (SEQ ID NO:180), YALDA (SEQ ID NO:181), YALDAR (SEQ ID NO:182), YALDARG (SEQ ID NO:183), GYAL (SEQ ID NO:184), GYALD (SEQ ID NO:185), GYALDA (SEQ ID NO:186), GYALDAR (SEQ ID NO:187), GYALDARG (SEQ ID NO:188), TGYAL (SEQ ID NO:189), TGYALD (SEQ ID NO:190), TGYALDA (SEQ ID NO:191), TGYALDAR (SEQ ID NO:192), TGYALDARG (SEQ ID NO:193), LTGYAL (SEQ ID NO:194), LTGYALD (SEQ ID NO:195), LTGYALDA (SEQ ID NO:196), LTGYALDAR (SEQ ID NO:197) or LTGYALDARG (SEQ ID NO:198); or (b) an analogue of any of the foregoing sequences that differs in one or more substitutions, deletions, additions and/or insertions such that that ability of the analogue to modulate a desmoglein-mediated function is not substantially diminished. Linear peptides having such sequences may be modified at the N- and/or C-termini, as in the peptides Nxe2x80x94Ac-LTGYALDARG-NH2 (SEQ ID NO:199).
Within other specific embodiments, a modulating agent as provided herein may comprise: (a) one or more Desmoglein-3 CAR sequences selected from the group consisting of: RAL, RALN (SEQ ID NO:151), RALNA (SEQ ID NO:200), RALNAQ (SEQ ID NO:201), RALNAL (SEQ ID NO:202), RALNAQG (SEQ ID NO:203), RALNALG (SEQ ID NO:204), CRAL (SEQ ID NO:157), CRALN (SEQ ID NO:158), CRALNA (SEQ ID NO:205), CRALNAQ (SEQ ID NO:206), CRALNAL (SEQ ID NO:207), CRALNAQG (SEQ ID NO:208), CRALNALG (SEQ ID NO:209), TCRAL (SEQ ID NO:210), TCRALN (SEQ ID NO:211), TCRALNA (SEQ ID NO:212), TCRALNAQ (SEQ ID NO:213), TCRALNAL (SEQ ID NO:214), TCRALNAQG (SEQ ID NO:215), TCRALNALG (SEQ ID NO:216), ITCRAL (SEQ ID NO:217), ITCRALN (SEQ ID NO:218), ITCRALNA (SEQ ID NO:219), ITCRALNAQ (SEQ ID NO:220), ITCRALNAL (SEQ ID NO:221), ITCRALNAQG (SEQ ID NO:222), ITCRALNALG (SEQ ID NO:223); or (b) an analogue of any of the foregoing sequences that differs in one or more substitutions, deletions, additions and/or insertions such that that ability of the analogue to modulate a desmoglein-mediated function is not substantially diminished. Linear peptides having such sequences may be modified at the N- and/or C-termini, as in the peptides Nxe2x80x94Ac-ITCRALNAQG-NH2 (SEQ ID NO:224) and Nxe2x80x94Ac-ITCRALNALG-NH2 (SEQ ID NO:225).
Within other specific embodiments, a modulating agent as provided herein may comprise: (a) one or more Desmocollin-1 CAR sequences selected from the group consisting of: YAT, YATT (SEQ ID NO:226), YATTA (SEQ ID NO:227), YATTAD (SEQ ID NO:228), YATTADG (SEQ ID NO:229), GYAT (SEQ ID NO:230), GYATT (SEQ ID NO:231), GYATTA (SEQ ID NO:232), GYATTAD (SEQ ID NO:233), GYATTADG (SEQ ID NO:234), AYAT (SEQ ID NO:235), AYATT (SEQ ID NO:236), AYATTA (SEQ ID NO:237), AYATTAD (SEQ ID NO:238), AYATTADG (SEQ ID NO:239), YGYAT (SEQ ID NO:240), YGYATT (SEQ ID NO:241), YGYATTA (SEQ ID NO:242), YGYATTAD (SEQ ID NO:243), YGYAT TADG (SEQ ID NO:244), YAYAT (SEQ ID NO:245), YAYATT)(SEQ ID NO:246), YAYATTA (SEQ ID NO:247), YAYATTAD (SEQ ID NO:248), YAYATTADG (SEQ ID NO:249), LYGYAT (SEQ ID NO:250), LYGYATT (SEQ ID NO:251), LYGYATTA (SEQ ID NO:252), LYGYATTAD (SEQ ID NO:253), LYGYATTADG (SEQ ID NO:254), LYAYAT (SEQ ID NO:255), LYAYATT (SEQ ID NO:256), LYAYATTA (SEQ ID NO:257), LYAYATTAD (SEQ ID NO:258), LYAYATTADG (SEQ ID NO:259), VYGYAT (SEQ ID NO:260), VYGYATT (SEQ ID NO:261), VYGYATTA (SEQ ID NO:262), VYGYATTAD (SEQ ID NO:263), VYGYATTADG (SEQ ID NO:264), VYAYAT (SEQ ID NO:265), VYAYATT (SEQ ID NO:266), VYAYATTA (SEQ ID NO:267), VYAYATTAD (SEQ ID NO:268), VYAYATTADG (SEQ ID NO:269), IYGYAT (SEQ ID NO:270), IYGYATT (SEQ ID NO:271), IYGYATTA (SEQ ID NO:272), IYGYATTAD (SEQ ID NO:273), IYGYATTADG (SEQ ID NO:274), IYAYAT (SEQ ID NO:275), IYAYATT (SEQ ID NO:276), IYAYATTA (SEQ ID NO:277), IYAYATTAD (SEQ ID NO:278) or IYAYATTADG (SEQ ID NO:279); or (b) an analogue of any of the foregoing sequences that differs in one or more substitutions, deletions, additions and/or insertions such that ability of the analogue to modulate a desmocollin-mediated function is not substantially diminished. Linear peptides having such sequences may be modified at the N- and/or C-termini, as in the peptides Nxe2x80x94Ac-LYGYATTADG-NH2 (SEQ ID NO:280) Nxe2x80x94Ac-LYAYATTADG-NH2 (SEQ ID NO:281) Nxe2x80x94Ac-VYGYATTADG-NH2 (SEQ ID NO:28 2) Nxe2x80x94Ac-VYAYATTADG-NH2 (SEQ ID NO:283) Nxe2x80x94Ac-IYGYATTADG-NH2 (SEQ ID NO:284) and Nxe2x80x94Ac-IYAYATTADG-NH2 (SEQ ID NO:285).
Within other specific embodiments, a modulating agent as provided herein may comprise: (a) one or more Desmocollin-2 CAR sequences selected from the group consisting of: FAT, FATT (SEQ ID NO:286), FATTP (SEQ ID NO:287), FATTPD (SEQ ID NO:288), FATTPDG (SEQ ID NO:289), AFAT (SEQ ID NO:290), AFATT (SEQ ID NO:291), AFATTP (SEQ ID NO:292), AFATTPD (SEQ ID NO:293), AFATTPDG (SEQ ID NO:294), IAFAT (SEQ ID NO:295), LAFATT (SEQ ID NO:296), IAFATTP (SEQ ID NO:297), IAFATTPD (SEQ ID NO:298), IAFATTPDG (SEQ ID NO:299), IIAFAT (SEQ ID NO:300), IIAFATT (SEQ ID NO:301), IIAFATTP (SEQ ID NO:302), IIAFATTPD (SEQ ID NO:303), IIAFATTPDG (SEQ ID NO:304), LIAFAT (SEQ ID NO:305), LIAFATT (SEQ ID NO:306), LIAFATTP (SEQ ID NO:307), LIAFATTPD (SEQ ID NO:308), LIAFATTPDG (SEQ ID NO:309); or (b) an analogue of any of the foregoing sequences that differs in one or more substitutions, deletions, additions and/or insertions such that that ability of the analogue to modulate a desmocollin-mediated function is not substantially diminished. Linear peptides having such sequences may be modified at the N- and/or C-termini, as in the peptides Nxe2x80x94Ac-IIAFATTPDG-NH2 (SEQ ID NO:310) and Nxe2x80x94Ac-LIAFATITDG-NH2 (SEQ ID NO:311).
Within other specific embodiments, a modulating agent as provided herein may comprise: (a) one or more desmocollin-3 or desmocollin4 CAR sequences selected from the group consisting of: YAS, YAST (SEQ ID NO:312), YASTA (SEQ ID NO:313), YASTAD (SEQ ID NO:314), YASTADG (SEQ ID NO:315), AYAS (SEQ ID NO:316), AYAST (SEQ ID NO:317), AYASTA (SEQ ID NO:318), AYASTAD (SEQ ID NO:319), AYASTADG (SEQ ID NO:320), IAYAS (SEQ ID NO:321), IAYAST (SEQ ID NO:322), IAYASTA (SEQ ID NO:323), IAYASTAD (SEQ ID NO:324), IAYASTADG (SEQ ID NO:325), LIAYAS (SEQ ID NO:326), LIAYAST (SEQ ID NO:327), LIAYASTA (SEQ ID NO:328), LIAYASTAD (SEQ ID NO:329), LIAYASTADG (SEQ ID NO:330); or (b) an analogue of any of the foregoing sequences that differs in one or more substitutions, deletions, additions and/or insertions such that that ability of the analogue to modulate a desmocollin-mediated function is not substantially diminished. Linear peptides having such sequences may be modified at the N- and/or C-termini, as in the peptides Nxe2x80x94Ac-LIAYASTADG-NH2 (SEQ ID NO:331).
Within other embodiments, a modulating agent comprises a dsc or dsg CAR sequence that is present within a cyclic peptide. Such cyclic peptides may have the formula: 
wherein W is a tripeptide selected from the group consisting of NQK, NRN, NKD, EKD, ERD, RAL, YAL, YAT, FAT, and YAS; wherein X1, and X2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein X1 and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within X1 and X2 ranges from 1 to 12; wherein Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2; and wherein Z1 and 4 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds.
Within other aspects of the present invention, polynucleotides encoding a modulating agent as described above are provided, along with expression vectors comprising such a polynucleotide and host cells transformed or transfected with such an expression vector.
The present invention further provides modulating agents that comprise an antibody or antigen-binding fragment thereof that specifically binds to a dsg or dsc CAR sequence as described above and modulates a desmosomal cadherin-mediated function.
Within further aspects, the present invention provides modulating agents comprising a non-peptide mimetic of any one of the desmosomal cadherin CAR sequences provided above.
In certain embodiments, a modulating agent may be linked to a drug, a detectable marker, a targeting agent and/or a support material. Modulating agents may also, or alternatively, comprise one or more of: (a) a cell adhesion recognition sequence other than a dsc or dsg CAR sequence; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a CAR sequence other than a dsc or dsg CAR sequence. For example, such an adhesion molecule may be a classical cadherin, nonclassical cadherin, integrin, occludin, claudin, N-CAM, fibronectin, laminin or other extracellular matrix protein.
Within further aspects, pharmaceutical compositions are provided, comprising a modulating agent as described above, in combination with a pharmaceutically acceptable carrier. Such compositions may additionally comprise a drug and/or one or more of: (a) a peptide comprising a cell adhesion recognition sequence other than a dsc or dsg CAR sequence; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a CAR sequence other than a dsc or dsg CAR sequence.
The present invention further provides, within other aspects, methods for modulating one or more desmosomal cadherin-mediated functions. Such methods generally comprise contacting a dsg- or dsc-expressing cell with a modulating agent as described above. Suitable cells include, but are not limited to, epithelial cells, endothelial cells and tumor cells. Within such methods, the modulating agent may, but need not, be present within a pharmaceutical composition as recited above.
The present invention further provides, within other aspects, methods for modulating cell adhesion, comprising contacting a dsc- and/or dsg-expressing cell with a modulating agent or a pharmaceutical composition as described above. Such modulating agents and compositions may inhibit or enhance cell adhesion.
Within other aspects, the present invention provides methods for inhibiting adhesion of dsc- and/or dsg-expressing cells in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits dsc- and/or dsg-mediated cell adhesion.
Within further aspects, the present invention provides methods for enhancing the delivery of a drug through the skin of a mammal, comprising contacting epithelial cells of a mammal with a drug and a modulating agent as described above, wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of the drug across the epithelial cells, and wherein the modulating agent inhibits dsg- or dsc-mediated cell adhesion. Such modulating agents may pass into the blood stream of the mammal. Within certain embodiments, the modulating agent is linked to the drug. The step of contacting may, but need not, be performed via a skin patch comprising the modulating agent and the drug, and such skin patches are further provided herein.
Methods are further provided for facilitating blood sampling in a mammal, comprising contacting epithelial cells of a mammal with a modulating agent as described above, wherein the modulating agent inhibits dsg- or dsc-mediated cell adhesion, and wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of one or more blood components across the epithelial cells. The step of contacting may be performed via a skin patch comprising the modulating agent, and (optionally) a reagent for detecting a blood component of interest, and such kits are specifically provided herein. Within certain embodiments, the epithelial cells are skin cells or are gum cells.
Within further aspects, methods are provided for enhancing the delivery of a drug to a tumor in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits dsg- or dsc-mediated cell adhesion. Suitable tumors include, but are not limited to, bladder tumors, ovarian tumors, breast tumors, stomach tumors and kidney tumors, and the modulating agent may be administered locally to the tumor or may be administered systemically.
Within other aspects, the present invention provides methods for treating cancer in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits dsg- or dsc-mediated cell adhesion. The mammal may be afflicted with a cancer such as a carcinoma, leukemia or melanoma, and the modulating agent may be administered to a tumor or systemically.
The present invention further provides, within other aspects, methods for inducing apoptosis in a desmosomal cadherin-expressing cell, comprising contacting a desmosomal cadherin-expressing cell with a modulating agent as described above, wherein the modulating agent inhibits desmosomal cadherin-mediated cell adhesion.
Within other aspects, the present invention provides methods for enhancing adhesion of desmosomal cadherin-expressing cells, comprising contacting dsg- or dsc-expressing cells with a modulating agent as described above, wherein the modulating agent enhances dsg- or dsc-mediated cell adhesion, wherein the step of contacting is performed under conditions and for a time sufficient to detectably enhance adhesion of the cells. Within certain embodiments, modulating agents for use within such methods are linked to a support molecule or a solid support.
Within related aspects, the present invention provides methods for facilitating wound healing and/or reducing scar tissue in a mammal, comprising contacting a wound in a mammal with a modulating agent as described above, wherein the modulating agent enhances desmosomal cadherin-mediated cell adhesion. Within certain embodiments, modulating agents for use within such methods are linked to a support molecules or a solid support.
Methods are also provided, within other aspects, for enhancing adhesion of foreign tissue implanted within a mammal, comprising contacting a site of implantation of foreign tissue in a mammal with a modulating agent as described above, wherein the modulating agent enhances dsg- or dsc-mediated cell adhesion. Such foreign tissue may be a skin graft or organ implant. Within certain embodiments, modulating agents for use within such methods are linked to a support molecules or a solid support.
Within further aspects, methods are provided for treating an autoimmune blistering disorder in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent enhances desmosomal cadherin-mediated cell adhesion. Within certain embodiments, such an agent may be administered topically to a blister. Modulating agents for use within such methods may be linked to a support molecule or a solid support.
The present invention further provides methods for detecting the presence of desmosomal cadherin-expressing cells in a sample, comprising: (a) contacting a sample with an antibody or antigen-binding fragment thereof that binds to a dsg or dsc CAR sequence as described above under conditions and for a time sufficient to allow formation of an antibody-cadherin complex; and (b) detecting the level of antibody-cadherin complex, and therefrom detecting the presence of desmosomal cadherin-expressing cells in a sample. The antibody may be linked to a support material or a detectable marker such as a fluorescent marker. In certain embodiments, the step of detecting is performed using fluorescence activated cell sorting.
Kits for detecting the presence of dsg- or dsc-expressing cells in a sample are also provided. Such kits may comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to a desmosomal cadherin CAR sequence; and (b) a detection reagent.
Within other aspects, the present invention provides methods for identifying a compound capable of modulating a desmosomal cadherin-mediated function, comprising: (a) contacting an antibody or antigen-binding fragment thereof that specifically binds to a dsg or dsc CAR sequence as described above with a test compound; and (b) detecting the level of antibody or fragment that binds to the test compound, and therefrom identifying a compound capable of modulating desmosomal cadherin-mediated cell adhesion.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.